JPH04501060A - A method for producing L-phenylacetyl carbinol (PAC), a microorganism for use in the method, and a method for preparing the microorganism - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] L ~ Phenylacetyl carbinol In order to follow the law, io'I for 11 坏 and 1 amount The present invention provides an intermediate in the production of 1-ephedrine and d-pseudoephedrine. Regarding the production method of phenylacetyl carbinol (PAC), which is useful as a body. Ru. The invention is also particularly suitable for use in the production of phenylacetyl carbinol. It also relates to adapted microorganisms and methods of feeding the microorganisms.

Pseudoephedrine and ephedrine are two main types of medicine. pseudof Aedrin is useful as a nasal decongestant and is used as a cough and sneeze cap. Cells, anti-inflammatory drugs, heavy-duty sprays, anti-inflammatory drops, and medicines for allergies and hay fever It is found as a component in Ephedrine is a topical decongestant, Useful in the treatment of mild forms of shock (CNS stimulant) and as a bronchodilator be.

L-ephedrine is a natural product found in many plant species. L-F The edrin is dried by an initial treatment with alkali followed by extraction with an organic solvent. Obtained from harvested plant material. d-pseudoephedrine is also found naturally However, it can be obtained in high yield from 1-ephedrine by Welsh rearrangement. It will be done.

L-phenylacetyl carbinol (PAC) is used in the synthesis of 1-ephedrine. It is a key intermediate for L-(-)phenylacetyl by Brewer's enzyme The conversion of benzaldehyde to carbinol was described by Newberg and Hir. sch, Bioche++, Z., 115:282-310 (1921). listed at the beginning. More specifically, benzaldehyde can be prepared as follows: 2P that can be converted to L-(-)phenylacetylcarbinol by fermentative yeasts Yeast conversion of benzaldehyde to produce AC and 1-ephedrine A combination of chemical transformations of PAC to listed in the issue. PAC was dissolved in optically pure ephedrine as follows: Can be converted by chemical reductive amination with thylamine: then 1-ephedo Phosphorus can be converted to d-pseudoephedrine in high yield as follows: Fermentation to form L-(-)phenylacetylcarbinol with high yield and purity. Microbiological conversion of benzaldehyde by the mother is important for commercial manipulation of synthetic routes It is clear from this reaction scheme that this is the case.

Traditional methods for the production of PAC from yeast include molasses, beer wort, Mg5O and other salts at a po of 5.5 to 6.0. first time Mixing of acetaldehyde and benzaldehyde after a short period of stirring and aeration Add some things. A final concentration of PAC of approximately 7.5 g/L was applied continuously for 5 to 10 hours. obtained by stirring and aeration. brewer or baker's yeast Dyl alcohol is always observed as a co-product. benzaldehyde-based The highest reported yield of PAC is about 73%. The remaining benzaldehyde is al Converted to call. Although acetaldehyde is not essential for PAC production, However, the addition of this compound is required to achieve the highest yield of PAC.

Most of the literature on the synthesis of PACs by fermentative yeasts discusses yield optimization. . The general requirement for high levels of yeast to obtain relatively low levels of PAC There is an agreement.

The available literature indicates that current yeast conversion methods of benzaldehyde to PAC are insufficient. Yes, suggesting that yeast productivity is low. In yeast, PAC production depends on substrates and It cannot be used in large amounts as it is degraded by increased exposure to the final product.

Additionally, current yeast conversions provide only low concentrations of PAC in the fermentation fluid. this is, Requires large process volumes and therefore large amounts of extraction solvent, and this is labor and adversely affects utility costs in commercial operations.

Furthermore, the yield of PAC from benzaldehyde is an undesired by-product, Benzyl by alcohol dehydrogenase to form benzyl alcohol It is lowered as a result of catalytic reduction of aldehydes. All of the PAC-producing strains tested produces benzyl alcohol.

Therefore, an improved method for producing PAC by yeast transformation of benzaldehyde is proposed. A need exists in the art for a method. This method is more effective than ever before. Gives higher yeast productivity and higher maximum concentration of PAC to the fermentation fluid. moreover , the catalytic reduction of benzaldehyde to benzyl alcohol should be minimized. Ru.

1 ri■! n The present invention provides an improved method for the production of PAC by yeast transformation of benzaldehyde. It is our objective to meet these needs in the industry by providing a method. It is. The method of the present invention provides a higher concentration of PAC in the fermentation fluid than in current methods. Allows for lower yeast productivity and higher concentrations to be obtained.

More specifically, the present invention provides a method for producing L-phenylacetyl carbinol (PAC). A method is provided, wherein the method comprises microorganisms in an aqueous fermentation medium containing assimilable carbon. providing an impregnated culture of the organism. Microorganisms are present in the fermentation medium at least benzaldehyde and pyruva to form PAC at a concentration of approximately 1.0 g/L. cultured under anaerobic or oxygen-limited conditions in the presence of The microorganism is Satsukaro Sudden outbreak of parent stock of Myces cerevisiae Mutants or mutants of the parent strain of Candida flareri It is a natural mutant. The mutant is more resistant to aldehyde inhibition and The corresponding strain using the parent strain does not produce acetaldehyde and under the same conditions. Provides a higher concentration of PAC in the fermentation medium than in the fermentation. PAC is separated from fermentation medium can be done.

The invention also provides methods for producing mutant microorganisms. The method is , when cultured with benzaldehyde and pyruvate in an aqueous medium. can produce measurable amounts of L-phenylacetyl carbinol (PAC). the method comprises supplying a microorganism that can kill the microorganism, wherein the microorganism is selected from species of Candida cerevisiae or Candida. Microorganisms are Chemically mutagenized by chemical mutagens or radiation. Obtained sudden change Metainduced microorganisms form colonies resistant to acetaldehyde cultured in the presence of acetaldehyde under conditions for Yeast cells are colonies It was isolated from Tested for the production of monophenylacetyl carbinol. High level PAC Cells from colonies that produce can be used in large-scale fermentations.

The present invention provides a similar method in which the mutagenized organism is under conditions to form colonies resistant to dione or PAC-dione. Cultivated in the presence of any of these substances. Yeast cells were isolated as described above. and tested.

Finally, the present invention provides a method for culturing with benzaldehyde and pyruvate in aqueous media. The growth of mutant microorganisms that can produce high levels of PAC when cultivated Provides physically pure cultures. Here, the culture is Identified as Ae P2180 1A-8pa and Sakonohisa Furareli dgr . The present invention utilizes these strains that are capable of producing high levels of PAC under the conditions described above. including mutants and variants of.

Mold plate■Gouisei desu plate The invention will be better understood by the following drawings: Figure 1 shows S. cerevisiae. Benzaldehyde/pills using the parent strains of E. and some of their mutant strains. Phenylacetyl carbinol (P) as a function of time in fermentation Figure 2 is a graph showing the concentration of S. cerevisiae and its parent strain; Benzaldehyde/pyruvate fermentation oven using several mutant strains of et al. is a graph showing acetaldehyde concentration as a function of time; Figure 3 shows C. Glue showing the concentration of PAC as a function of time for strains of H. fraleri Rough; Figure 4 shows the effects on the rate and extent of PAC fermentation using the K. A graph comparing the effects of Drin; and Figure 5 shows PAC fermentation using the wild type strain and three mutant strains of ζ Yui and Fumi. 2 is a graph comparing speed and volume.

The story of a good news The method of the present invention provides high yields of L-phenylacetylcarbinocarbons by microbiological transformation. (PAC). The abbreviation "PAC" stands for L-(-) of phenylacetyl carbinol identified as phenylacetyl carbinol. Used to refer to stereospecific forms. Name L-(-)phenylacetylka Rubinol is used interchangeably with the name 1-phenylacetylcarbinol. , and both names are abbreviated as PAC'.

PAC is prepared by conversion of benzaldehyde and pyruvate. ′Pilvay "" is used in its conventional sense when referred to as an ingredient.

The conversion of benzaldehyde and pyruvate is carried out according to the present invention by mutant strains of microorganisms. done in a method. As used in the present invention, the term "mutant" refers to the parent Includes all progeny of the parent organism in which genotypic differences exist between the strain and its progeny It means to do. The term also refers to It is also meant to include progeny that are phenotypically different from the parent strain. of course, The term further encompasses progeny that exhibit both genotypic and phenotypic differences from the parent line. Contains.

More specifically, the method of the present invention involves combining pyruvic acid and benzaldehyde with Yeast seeds effectively contain the enzyme pyruvate decarboxylate to nyl acetyl carbinol. can produce silase and therefore endogenous pyruvate decarboxylase. including As used in the present invention, the abbreviation “PDC,” refers to the enzyme pilve means todecarboxylase.

Pyruvate decarboxylase catalyzes the conversion of benzaldehyde to PAC . This enzyme can also convert pyruvate to acetaldehyde. a The formation of cetaldehyde is evident in the decreased yield of PAC from benzaldehyde. It is thought to inhibit the enzyme responsible for this. The present invention is resistant to aldehyde inhibition. We supply mutant strains with sexual characteristics. According to this, compared to the parent strain, the fermentation medium The abrupt change in benzaldehyde conversion as evidenced by the This means that there is reduced inhibition of the activity of the mutant strain. PAC in fermentation medium When two fermentations carried out under the same conditions are compared, the concentration of It is higher in case of mutant strain than in case of case. In the preferred Mr. M of the present invention, The mutant strain produces less acetaldehyde from benzaldehyde than the parent strain. .

Mutations were selected from species of S. cerevisiae or C. fraleri. induced by selected microorganisms. The resulting mutant is susceptible to acetaldehyde inhibition. Cultured in the presence of acetaldehyde under conditions that form colonies resistant to be nourished. Cells from that colony were isolated and treated with benzaldehyde and pinol. The yield of PAC in fermentation with rubate is tested. Cells also The yield of cetaldehyde is also tested. Therefore, a high level of PAC generation and select yeast cells from colonies that produce acetaldehyde at low levels. and these microorganisms to produce PAC with improved yield compared to the parent strain. It is possible to use objects. PAC at higher yields in commercial operations The production of is particularly advantageous since the cost of production will be reduced.

A method for preparing yeast mutant strains with induced mutations is first described. Will. This was done by culturing the mutant for high levels of PAC production and Accompanied by an explanation of the conditions for selection. In the conversion of benzaldehyde to PAC A description of the conditions for using the mutants is provided next.

1. Wild-type yeast was chemically mutagenized. emitting substances such as methylnitrosoguanidine, nitrous acid or ethylmethanesulfone It can be triggered suddenly by triggering. A preferred chemical mutagen is N- Methyl-N'-nitro N-nitrosoguanidine (NTG). wild type yeast The mother can also be mutagenized by irradiation with ultraviolet light or ionizing radiation, e.g. gamma rays. can be done.

2. Acetaldehyde, pyruvate aldehyde, propionaldehyde, formal A compound that is resistant to dehydes, furfural, glyoxal and alpha-ketobutyric acid. Natural mutant strains can be prepared. More specifically, the active PAC-producing mutant strain It can be obtained as follows.

Using the microorganisms containing the induced mutations prepared as described above, Mutants resistant to acetaldehyde are The mutant strain can be cultured by placing the spread plate in a container. Wear. Acetaldehyde is volatile, so if the jar is sealed, its large Air is saturated with acetaldehyde. Resistant mutants proliferate under these conditions. to ensure that it will reproduce and colonize (the parent plant does not) , acetaldehyde from about 3504 to about 1000p! should be used in amounts of Ta For example, about 3,504 grams of acetaldehyde can be added to a 3.75 L jar.

Using this method, mutations in S, S. cerevisiae P-2180-IA [wild type (WT) haploid strain] with N-methyl-N'-nitro N-nitrosoguanidine. was induced. Because many mutants are resistant to pyruvate aldehyde chosen.

Mutants are identified in Table 1 below.

Obtained by treatment of P-2180-IA and regarding acetaldehyde resistance ``Suddenly'' Yutaka Strains resistant to other inhibitors can be cultivated using similar methods. Ta For example, the strain can be prepared using similar methods to produce 2,3-dihydroxybenzaldehyde. , 2,4-dihydroxybenzaldehyde, 2,5-dihydroxybenzalde Hyde, 3,4-dihydroxybenzaldehyde, furfural (2-formal presence of glyoxal, formaldehyde and propionaldehyde can be cultured under

If the aldehyde used is not volatile, it is preferably sealed. into the culture medium rather than being added to the jar.

3. Screening for aldehydes for PAC Assaying the effectiveness of each of the mutant yeast strains in producing PAC To achieve this, batch fermentation can be carried out as follows. fixed or free A known amount of cells is added to a buffer solution containing the required substrate. In particular, A suitable standard reaction mixture for the production of PAC in a reaction mixture consists essentially of the following components: : Potassium citrate buffer, pH 6,0100mM magnesium chloride (MgCl□ ) 1mM Polyethylene Glycol-1O00 (PEG-1000) 20% Pi Sodium rubate 110mM Benzaldehyde (technical grade) 110n+M Next, standard reaction mixture containing cells Shake at room temperature for several hours.

Cell samples are taken at various intervals and centrifuged. The obtained supernatant liquid was PA Quantitative analysis of the presence of C. The PAC assay uses Eggleton star e, Modified from Biochem, J, 37:526-529 (1943). Special In addition, Eggleton's method of boiling diacetylation by absorbance at 525n- This is for quantifying the compound. The PAC reading is the contribution of any of the absorbing species at 525 nw. amended by.

Each of the strains identified in Table 1 was treated with benzaldehyde and After fermentation with pyruvate and pyruvate, the concentration of PAC in the culture medium revealed that It was tested for its ability to produce PAC as described above. After 5 hours, fermentation medium Table 2 shows the concentrations of PAC (mM) for individual microorganisms. The results reported in EP-2180-IA and Table 2 are based on the mutant strain S, S. cerevisiae. Ae P-2180-14-8□ than the wild type (-D) strain P-2180-IA. Showing to be substantially more effective in producing PAC under the mentioned conditions . The improved performance of this mutant is due to the inhibition of PDC by aldehydes. This seems to be the result of a decline in

Using the parent strain S, S. cerevisiae P-2180-IA and several of its mutants. A benzaldehyde/pyruvate fermentation was carried out, and the fermentation medium was Samples were taken in individual cases after 3 and 5 hours of time.

Regarding Figure 1, all strains have a very rapid rate of PAC formation initially. As far as possible, they showed similar fermentation kinetic patterns. Subsequently, the slow rate of PAC formation and ultimately, the instability of PAC in the fermentation medium reduces the PAC concentration. There was a slight decrease in Mutant strain P-2180-IA-8.

is capable of giving the highest yield of PAC at each fermentation time for which measurements are carried out. This is clear from Figure 1. This mutant has a higher yield of PAC than its parent strain P Substantially superior to -2180-IA. Mutant strain P-2180-LA- 23p, and P-2180-16□ also outperformed the parent strain P-21 in yield of PAC. Superior to 80-LA.

Furthermore, mutant S, P. cerevisiae P-2180-IA-8P, Significantly lower acetaldehyde than its parent strain in aldehyde/pyruvate fermentation generate a code. In particular, Figure 2 shows the concentration of acetaldehyde (m M) is shown. The concentration of acetaldehyde depends on the individual reaction in which the medium is sampled. For the mutant P-2180-IA-8□ in time than for the parent strain P-2180 -It was high for IA. A decrease in acetaldehyde concentration was observed in mutant strain P- Causative factors in the observed increase in PAC production by 2180-IA-8, It is. Mutant S, (-Levisiae P-2180-IA-25,, Hama In addition, as is clear from Figure 2, it produced even lower acetaldehyde than the parent strain. .

The mutant strain P-2180-IA-L has no distinguishing properties from its parent strain. It has one characteristic. Pyruvate is good for the growth of S. cerevisiae tongue. It is well known that it is an unfavorable substrate. The mutant uses more carbon sources than the parent strain. Propagates well with pyruvate as a. Mutant P-2180-IA-8 □ grows well on pyruvate, and the parent strain grows well on pyruvate. This does not suggest that the conversion of the cells is responsible for growth inhibition. The mutant is that it Selected for aldehyde resistance, revealing the inhibitory effect of acetaldehyde I can escape.

Tolerance to the inhibitory effects of 2,3-dihydroxybenzaldehyde on cellular metabolism To illustrate, S. cerevisiae P-2180-IA and mutant P-2180 -IA-8,, respectively, were placed on the growth medium in culture blood. equal volume of the culture impregnated with aldehyde. Test the resulting cell colonies and mutagenize them The plant was found to exhibit a smaller area of inhibition (clear area) than the parent strain. child This indicates that S. cerevisiae P-2180-IA-8□ is more sensitive to this aldeline than its parent strain. It was shown to be more resistant to the inhibitory effects of Hyd. formaldehyde, glycerin Mutant P-2180-IA for oxal and 2-furaldehyde inhibition Similar results were obtained for the -8□ resistance.

In addition to strain P-2180-IA-8□, formaldehyde, 2,3-dihydroxy Mutations in S. cerevisiae for resistance to benzaldehyde and furfural Stock was obtained. Nothing is better at PAC production than the parent strain P-2180-LA. won.

A mutant of C. fulareri c1gr that is resistant to acetaldehyde is also described above. It was prepared as follows. Forming PAC from pyruvate and benzaldehyde As a result, none of these were superior to the parent strain.

Besides selecting mutants resistant to aldehydes, two other approaches , increase the amount of pyruvate decarboxylase in cells or change the characteristics of the enzyme. It was used to Culture and selection methods used for aldehyde tolerance Same method as above except agar plates were used instead of jars.

The first approach was to use analogs of the products of the pyruvate and benzaldehyde reactions. The aim was to select mutants resistant to resistant to ephedrine Mutants were selected. 2. Nine dgr Experiments designed to select ephedrine-resistant strains of Natural mutants C, Frareli dgr E, D and G were obtained. For individual strains, use the method above was assayed for PAC production using Figure 3 shows wild type strain (WT) and The concentrations of PAC (mM) obtained with the two mutants are shown. Mutant ζ− 2 shi kJ ~ dgr　H (DGRE) is substantially more effective than the WT strain in PAC production. It was found to be outstanding. Final concentration of PAC and P in the fermentation medium Both rates of AC formation were outstanding for mutant DGR-H. On the other hand, The mutant ζl-starter dgrF (DGRF) has a lower favorable outcome than the WT strain. gave fruit. The results in Figure 3 highlight the need for caution in selecting appropriate mutant strains. show.

Mutant DGR-E shows that ephedrine in the fermentation medium does not affect PAC production. Further tests were conducted to determine if any effects were present. Rate and extent of PAC production degree is found to be superior when the mutant DGR-ε is compared with the WT strain. Served. The results are shown in FIG. Ephedrine (+) in fermentation medium eph) inhibits PAC formation by both the mutant DGR-E and the WT strain. However, the mutant strain gave less effect than the parent strain. In Figure 4 The results also confirm the results in Figure 3, i.e. mutant DGR-E In the absence of ephedrine in the fermentation medium (-eph), W in PAC production Superior to T stock.

FIG. 5 shows that two other isolates, dgr D and dgr G, are shows significantly superiority over the parent strain in the absence of phosphorus.

S. cerevisiae mutants resistant to ephedrine have outstanding PAC No producers were given.

The second approach is to use PAC-diones, another PAC analogue. The aim was to select mutants of Σ-dir for resistance to Σ. P AC-dione has the following general formula: PAC-dione resistant isolates produce more PAC than the parent strain in the presence of dione, but , did not excel in Zeon's absence.

We also selected mutants of S. cerevisiae that were resistant to PAC-diones; These were synthesized from the parent strain by synthesis of PAC from pyruvate and benzaldehyde. was also not good.

4. Benzal-human and pyruvate... Prepare PAC in aqueous fermentation medium using the mutant strain prepared as above. The method of the invention can be carried out in a conventional bioreactor. The resulting zero fermentation is preferably carried out in a stirred tank reactor. Mixed but substantially impeller, agitator or other device to keep the container contents in a homogeneous form may be provided by other suitable means for maintenance.

The method of the invention is carried out by impregnation culture of mutant strains and substantially anoxic or anaerobic culture. It can be done under certain conditions. Freely moving cells or fixed cells can be used .

Substrate to grow yeast cells and increase biomass concentration in the fermenter can be added to the fermenter at any time. Conventional organic fermentable carbon substrates, such as carbohydrates, Get used for the purpose. Glucose is a preferred substrate. Organic substrate alone It can be used with or with other organic substrates.

The carbon substrate can be fed to the fermentor in an aqueous solution. The concentration of substrate in the fermentation medium depends on the culture conditions. It will depend on the case. In any case, carbon substrates are useful for cell growth and cell maintenance. used in sufficient quantities for Contains about 50 g/L to about 100 g/L glucose It has been found that aqueous solution 1 is suitable as a carbon source.

Yeast cells for fermentation 21/a are placed in a reaction vessel, such as a shake flask or a fermenter. 0 different cultures that can be grown on nutrient media containing a source of assimilable carbon A nutrient medium may be used.

Complex media found to be suitable for use in fermentation are listed in Table 3 below. be done.

Glucose 100g/L Yeast extract 6 g/L (NH*)zsOa 4 g/L MgSO,・IHto 0.6g/LKHzPOa 1g/L Cell growth is typically carried out at 30"C and 5.5 p)l in a reaction vessel. The biomass concentration during the growth phase can be determined by sampling the container contents. , and can be monitored by measuring the optical density (OD) of the sample. . The optical density is about 5 to about 35 as measured by absorbance at 660n. cells should be made oxygen-limited or anaerobic, or both. cells are group The course should be harvested before it is exhausted. Cell harvest was carried out at 4-10°C. This can be done by centrifugation.

While characterizing mutant microorganisms, a distinct degree of variability in PAC production was observed. It will be done. The rate of PAC formation is correlated with the growth phase of the culture used in its generation method. It was found that this is related. In particular, in cell cultures used for PAC production. It was found that the level of dissolved oxygen in the It was. Furthermore, if the culture enters stationary phase due to depletion of some nutrients other than oxygen, PAC synthesis was found to be severely limited. However, the actual culture The density of does not seem to have a major behavior on the fermentation reaction. growth curve Sampling of cultures along (early, middle or late logarithmic growth phase to stationary phase) is similar, as long as the cells are grown anaerobically for a period of time before being used in the PAC reaction. yield similar results. For example, this is glucose (50g/L) This can be achieved by growing microorganisms in fermenters on complex media containing . When the biomass reaches an optical density of about 30, the aerobic reaction is stopped and The medium is allowed to be depleted of oxygen. The cells do not consume glucose and 6~ Grow anaerobically for 16 hours. To harvest, soak the culture in ice. pump through a cooled cooling coil and remove the cells by centrifugation. be able to.

The benzaldehyde used in the practice of this invention is generally commercially available from the technical or It is a medicinal variety. Pyruvate is usually made from pyruvate or its non-toxic water-soluble salts. derived from technical or medicinal varieties. Non-toxic alkali metal salts, such as sodium pyruvate Thorium is preferred.

Pyruvate and benzaldehyde should be dispersed evenly through the biomass in the reaction medium. may be added individually to the reaction if sufficiently stirred to ensure . Main departure In a preferred embodiment, pyruvate and benzaldehyde are combined in an aqueous medium. and the resulting composition is added to the reaction.

At the start of the fermentation reaction, the concentration of benzaldehyde in the fermentation medium is generally about 5 g. /L to about 20 g/L, preferably about 12 g/L to about 15 g/L. similarly , the concentration of pyruvate in the fermentation medium is about 1:1 to about 20 g/L at the start of fermentation, Preferably it is about 12 g/L to about 15 g/L.

The benzaldehyde:pyruvate weight ratio in the fermentation medium at the start of fermentation is typically from about 0.5:1 to about 2:1, preferably from about 1:1 to about 1.2:1.

The chemical components in the nutrient medium and the amounts of individual components are important for cell mass production. should be sufficient to meet the elemental requirements, and during PAC generation, the synthesis Adequate energy should be provided for growth and maintenance.

The fermentation process is carried out over a moderate temperature range.

The fermentation temperature is generally about 15°C to about 30°C, preferably about 0°C to about 22°C. It is. The optimum fermentation temperature depends on the microorganism used, and the optimum temperature It can be determined by experiment.

The process of the invention can also be carried out over a range of medium pH values in the culture medium. I can. The pH will generally be about 5 to about 8, preferably about 6 to about 6.5. . The pH in the culture medium can decrease or increase during fermentation. non-toxic p Hm conditioners or buffers may be added to the fermentation medium as needed.

The method of the invention is performed under sufficient sterile conditions to ensure cell viability and metabolism. be called. This requires careful selection of microorganisms, sterilization of equipment used for fermentation. and requires possible sterilization of reagents.

Fermentors are operated over a wide range of biomass concentrations, and the optimum concentration is can be determined without experimentation. As mentioned above, the biomass concentration is It does not appear to have a major behavior towards fermentation reactions. The actual range of that value is It will generally depend on the economics of the method.

The concentration of PAC in the fermentation medium is maximized to reduce the cost of product recovery. Should. The method of the invention provides for at least about 1 g/L PAC in the fermentation medium. at a concentration, and preferably at a PACI degree of at least about 10 g/L, and especially about 1 Concentrations from 5°C to about 15 g/L can be carried out.

The process of the invention can be carried out on a batch or continuous basis. By continuous fermentation For the production of PAC, microorganisms are placed in a column and a standard reaction mixture is added to it. column. Thiamine pyrophos, a cofactor for the enzyme PDC□ Phate (TPP) is included in the standard reaction mixture, for example at a concentration of 0.1 wM. obtain. Both aliquots of column effluent were collected and quantitatively sampled for the presence of PAC. Google.

PACs can be recovered from fermentation media using conventional techniques.

For example, biomass is removed from the liquid phase in fermentation media by filtration, centrifugation or sedimentation. can be recovered from. The resulting liquid phase is further treated to a-condense the PAfJ liquid. can be managed. PAC can be removed from its solution by solvent extraction.

PAC Neuberg, Biochem, Z, 128:610 (1922) It can be purified according to the method of GC/MS profile of the purified product indicates the parent peak with a molecular weight of 150. The products of the reaction also have the correct optical The optical rotation needle shows that the isomer is L-phenylacetyl carbinol. measured. Conversion to ephedrine takes place by reaction with methylamine, And the results were proven.

More details on the methods that can be used to generate PACs using the mutant strains of the invention. New explanation: Robert J, 5eeiy, mouthona1dL, Heefner , Robert ν, Hagea+an, Michael J., Yarus and 5ally 8, 5ullivan - (Attorney Docket No. 5YNE-037) and °゛LL-phenylacetyl carbon A method for producing Rubinol AC), a fixed cell mass for use in the method, and U.S. Pat. Included. The entire disclosure of this application is dependent on and includes the references herein. incorporated by.

In summary, the present invention provides a method for microbiological conversion of benzaldehyde to PAC. An effective method and microorganisms for use in the method are provided. About PAC The productivity of microorganisms is high. Furthermore, during its conversion, it eliminates the formation of acetaldehyde. However, it is possible to obtain relatively low concentrations of PAC in the fermentation fluid.

FIG, / time (hour) to Ya Special Table Sen4-501060 (9) time (hour) FIG. 4 time (hour) international search report 1 M”〒/n1ll#1phrase/nmumufu1

Claims (1)

[Claims] 1. A method for producing L-phenylacetyl carbinol (PAC), comprising: providing an aqueous fermentation medium comprising an impregnated culture of microorganisms and a source of assimilable carbon; Benz to form a PAC in the fermentation medium at a concentration of at least about 1 g/h. The microorganisms are grown under anaerobic or oxygen-limited conditions in the presence of aldehydes and pyruvate. comprising culturing; and The microorganism is a mutant of the parent strain of Saccharomyces cerevisiae or Candida It is a mutant of the parent strain of Fraleri, and the mutant inhibits aldehyde. and than the corresponding fermentation using the parent strain under the same conditions. Rimo fermentation medium produces lower acetaldehyde and higher concentration of PAC A method characterized by: 2. The parent strain is S. cerevisiae P-2180-IA, and the sudden The mutant is S. cerevisiae mutant strain P-2180-IA-2pa, P-218 0-1A-3pa, P-2180-IA-4pa, P-2180-IA-6pa , P-2180-IA-7pa, P-2180-IA-8pa, P-2180- IA-9pa, P-2180-IA-10pa, P-2180-IA-11pa , P-2180-IA-12pa, P-2180-IA-13pa, P-218 selected from the group consisting of 0-IA-14pa and P-2180-IA-16pa. The method according to claim 1. 3. The parent strain is S. cerevisiae P-2180-IA, and the sudden The mutant is S. cerevisiae strain P-2180-IA-8pa The method described in 1. 4. The fermentation is carried out at a temperature of about 15°C to about 30°C in a fermentation medium having a pH of about 5 to about 8. 4. A method according to claim 3, which is carried out at temperature. 5. The method according to claim 4, wherein the fermentation is carried out by continuous stirring of the fermentation medium. Law. 6. At the start of fermentation, the fermentation medium contains about 5 g/L to about 20 g/L of benzaldehyde. from about 59 g/L to about 20 g/L, and the benzaldehyde Claim 5 wherein the de:pyruvate is present in a weight ratio of about 0.5:1 to about 2:1. Method described. 7. The concentration of PAC in the fermentation medium is about 12 g/L to about 15 g/L. The method described in scope item 6. 8. When cultured with benzaldehyde and pyruvate in an aqueous medium, Can produce high levels of 1-phenylacetyl carbinol (PAC) A method for producing a yeast microorganism comprising: (A) When cultured with benzaldehyde and pyruvate in an aqueous medium , provide a yeast microorganism capable of producing measurable amounts of PAC, and here the Is the mentioned microorganism a species of Saccharomyces cerevisiae or a species of Candida flareli? selected; (B) mutagenizing the microorganism with a chemical mutagen or irradiation; (C) the resulting mutagenized microorganism is reduced in acetaldehyde In the presence of acetaldehyde under conditions for forming cell colonies with inhibition (D) From colonies resistant to acetaldehyde inhibition. isolate the yeast cells; and (E) For the yield of PAC in fermentations involving benzaldehyde and pyruvate. testing yeast cells isolated from said colony. 9. Acetaldehyde yield in fermentations involving benzaldehyde and pyruvate further comprising testing the yeast cells isolated from said colony for a The method according to claim 8, comprising: 10. When cultured with benzaldehyde and pyruvate in aqueous media , can produce high levels of 1-phenylacetyl carbinol (PAC). 1. A method for producing a yeast microorganism that can (A) When cultured with benzaldehyde and pyruvate in an aqueous medium , providing a microorganism capable of producing a measurable amount of PAC, wherein said microorganism is The organism is selected from strains of the type Candida fraleri; (B) mutagenizing the microorganism with a chemical mutagen or irradiation; (C) making the resulting mutagenized microorganism resistant to ephedrine inhibition; Cultured in the presence of ephedrine under conditions to form sexually active colonies. (D) isolating cells from colonies that are resistant to ephedrine inhibition; and (E) For the yield of PAC in fermentations involving benzaldehyde and pyruvate. testing cells isolated from said colony. 11. of acetaldehyde in fermentations involving benzaldehyde and pyruvate. further comprising testing cells isolated from said colony for yield. 11. The method according to claim 10. 12. When cultured with benzaldehyde and pyruvate in aqueous media , can produce high levels of 1-phenylacetyl carbinol (PAC). 1. A method for producing microorganisms that can (A) When cultured with benzaldehyde and pyruvate in an aqueous medium , providing a microorganism capable of producing a measurable amount of PAC, wherein said microorganism is The organism is selected from strains of Candida flareli; (B) The microorganism is mutagenized with RuN′-nitro-N-nitrosoguanidine (N76); (C) The resulting mutagenized microorganisms were subjected to PAC-dione inhibition. Cultivation in the presence of PAC-dione under conditions to form resistant colonies (D) Cells were isolated from colonies resistant to PAC-dione damage. Release; and (E) For the yield of PAC in fermentations involving benzaldehyde and pyruvate. testing cells isolated from said colony. 13. of acetaldehyde in fermentations involving benzaldehyde and pyruvate. further comprising testing cells isolated from said colony for yield. 13. The method according to claim 12. 14 Biology of Saccharomyces cerevisiae P-2180-IA-8pa pure culture. 15. Biologically pure culture of Candida fulareri dgr.